Methods for enhancing the production of viral vaccines in cell culture

ABSTRACT

Methods for enhancing the production of viral vaccines in animal cell culture are described. These methods rely on the manipulation of the cellular levels of certain interferon induced antiviral activities, in particular, cellular levels of double-stranded RNA (dsRNA) dependent kinase (PKR) and 2′-5′ oligoadenylate synthetase (2-5A synthetase). In cell cultures deficient for PKR or 2-5A synthetase, viral yield is enhanced by several orders of magnitude over cell cultures with normal levels of these proteins making these cell cultures useful for the production of viral vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 09/113,016filed Jul. 9, 1998, which in turn is a continuation of U.S. Ser. No.08/700,198 filed Aug. 21, 1996, which claims benefit of ProvisionalApplication U.S. Ser. No. 60/002,621 filed Aug. 22, 1995. Each of thesecited Applications are hereby incorporated, in their entirety, byreference.

INTRODUCTION

[0002] 1. Technical Field

[0003] The present invention relates to methods for the production ofvirus for vaccine production in cell culture.

[0004] 2. Background

[0005] Effective control of influenza pandemics depends on earlyvaccination with the inactivated virus produced from newly identifiedinfluenza strains. However, for more effective pandemic control,improvements in the manufacturing and testing of the vaccine are needed.Influenza viruses undergo very frequent mutations of the surfaceantigens. Consequently, vaccine manufacturers cannot stock-pile millionsof doses for epidemic use. Current influenza control methods demandconstant international surveillance and identification of any newlyemergent strains coupled with vaccine production specific for the newlyidentified strains. Current influenza vaccine production, which requiresthe use of embryonated eggs for virus inoculation and incubation, iscumbersome and expensive. It can also be limited by seasonalfluctuations in the supply of suitable quality eggs. Thus, forproduction of mass doses of monovalent vaccine in a short time, it wouldbe advantageous to develop alternate, egg-independent productiontechnology. In this respect, production of an influenza vaccine on astable cell line may solve many of the problems in mass production.However, the yield of human influenza viruses on tissue culture isdisappointingly much lower than in embryonated eggs (Tannock et al.Vaccine 1985 3:333-339). To overcome these limitations and improve thequality of vaccines, it would be advantageous to develop cell culturelines which provide an enhanced yield of virus over those currentlyavailable.

[0006] In using mammalian cell lines for whole virion vaccineproduction, a common problem for vaccine manufacturers is that mammaliancells have intrinsic antiviral properties, specifically, the interferon(IFN) system, which interferes with viral replication. IFNs can beclassified into two major groups based on their primary sequence. Type Iinterferons, IFN-α and IFN-β, are encoded by a super family ofintronless genes consisting of the IFN-α gene family and a single IFN-βgene. Type II interferon, or IFN-γ, consists of only a single type andis restricted to lymphocytes (T-cells and natural killer cells). Type Iinterferons mediate diverse biological processes including induction ofantiviral activities, regulation of cellular growth and differentiation,and modulation of immune functions (Sen, G. C. & Lengyel, P. (1992) J.Biol. Chem. 267, 5017-5020; Pestka, S. & Langer, J. A. (1987) Ann. Rev.Biochem. 56, 727-777). The induced expression of Type I IFNs, whichinclude the IFN-α and IFN-β gene families, is detected typicallyfollowing viral infections. Many studies have identified promoterelements and transcription factors involved in regulating the expressionof Type I IFNs (Du, W., Thanos, D. & Maniatis, T. (1993) Cell 74,887-898; Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami,T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham,A., Potter, J., Furlonger, C. L., Narendran, A., Suzuki, H., Ohashi, P.S., Paige, C. J., Taniguchi, T. & Mak, T. W. (1993) Cell 75, 83-97;Tanaka, N. & Taniguchi, T. (1992) Adv. Immunol. 52, 263-81). However, itremains unclear what are the particular biochemical cues that signifyviral infections to the cell and the signaling mechanisms involved (fora recent review of the interferon system see Jaramillo et al. CancerInvestigation 1995 13:327-337).

[0007] IFNs belong to a class of negative growth factors having theability to inhibit growth of a wide variety of cells with both normaland transformed phenotypes. IFN therapy has been shown to be beneficialin the treatment of human malignancies such as Kaposi's sarcoma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma and hairy cell leukemia aswell as the treatment of infectious diseases such as papilloma virus(genital warts) and hepatitis B and C (reviewed by Gutterman Proc. NatlAcad Sci. 91:1198-1205 1994). Recently, genetically-engineeredbacterially-produced IFN-β was approved for treatment of multiplesclerosis, a relatively common neurological disease affecting at least250,000 patients in the US alone.

[0008] IFNs elicit their biological activities by binding to theircognate receptors followed by signal transduction leading to inductionof IFN-stimulated genes (ISG). Several of them have been characterizedand their biological activities examined. The best studied examples ofISGs include a double-stranded RNA (dsRNA) dependent kinase (PKR,formerly known as p68 kinase), 2′-5′-linked oligoadenylate (2-5A)synthetase, and Mx proteins (Taylor J L, Grossberg S E. Virus Research1990 15:1-26.; Williams B R G. Eur. J Biochem. 1991 200:1-11). Human MxA protein is a 76 kD protein that inhibits multiplication of influenzavirus and vesicular stomatitis virus (Pavlovic et al. (1990) J Viol. 64,3370-3375).

[0009] 2′-5′Oligoadenylate synthetase (2-5A synthetase) uses ATP tosynthesize short oligomers of up to 12 adenylate residues linked by2′-5′-phosphodiester bonds. The resulting oligoadenylate moleculesallosterically activate a latent ribonuclease, RNase L, that degradesviral and cellular RNAs. The 2-5A synthetase pathway appears to beimportant for the reduced synthesis of viral proteins in cell-freeprotein-synthesizing systems isolated from IFN-treated cells andpresumably for resistance to viral infection in vivo at least for someclasses of virus.

[0010] PKR (short for protein kinase RNA-dependent) is the onlyidentified double-stranded RNA (dsRNA)-binding protein known to possessa kinase activity. PKR is a serine/threonine kinase whose enzymaticactivation requires binding to dsRNA or to single-stranded RNApresenting internal dsRNA structures, and consequent autophosphorylation(Galabru, J. & Hovanessian, A. (1987) J Biol. Chem. 262, 15538-15544;Meurs, E., Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams,B. R. G. & Hovanessian, A. G. (1990) Cell 62, 379-390). PKR has alsobeen referred to in the literature as dsRNA-activated protein kinase,P1/e1F2 kinase, DAI or dsI for dsRNA-activated inhibitor, and p68(human) or p65 (murine) kinase. Analogous enzymes have been described inrabbit reticulocytes, different murine tissues, and human peripheralblood mononuclear cells (Farrel et al. (1977) Cell 11, 187-200; Levin etal. (1978) Proc. Natl Acad. Sci. USA 75, 1121-1125; Hovanessian (1980)Biochimie 62, 775-778; Krust et al. (1982) Virology 120, 240-246;Buffet-Janvresse et al. (1986) J Interferon Res. 6, 85-96). The bestcharacterized in vivo substrate of PKR is the alpha subunit ofeukaryotic initiation factor-2 (eIF-2a) which, once phosphorylated,leads ultimately to inhibition of cellular and viral protein synthesis(Hershey, J. W. B. (1991) Ann. Rev Biochem. 60, 717-755). PKR canphosphorylate initiation factor elF-2α in vitro when activated bydouble-stranded RNA (Chong et al. (1992) EMBO J. 11, 1553-1562). Thisparticular function of PKR has been suggested as one of the mechanismsresponsible for mediating the antiviral and antiproliferative activitiesof IFN-α and IFN-β. An additional biological function for PKR is itsputative role as a signal transducer. Kumar et al. demonstrated that PKRcan phosphorylate IκBα, resulting in the release and activation ofnuclear factor κB (NF-κB) (Kumar, A., Haque, J., Lacoste, J., Hiscott,J. & Williams, B. R. G. (1994) Proc. Natl. Acad. Sci. USA 91,6288-6292). Given the well-characterized NF-κB site in the IFN-βpromoter, this may represent a mechanism through which PKR mediatesdsRNA activation of IFN-β transcription (Visvanathan, K. V. &Goodbourne, S. (1989) EMBO J. 8, 1129-1138).

[0011] The catalytic kinase subdomain of PKR (i.e., of p68 (human)kinase and p65 (murine) kinase) has strong sequence identity (38%) withthe yeast GCN2 kinase (Chong et al. (1992) EMBO J. 11, 1553-1562; Fenget al. (1992) Proc. Natl. Acad. Sci. USA 89, 5447-5451). Recombinant p68kinase expressed in yeast Saccharomyces cerevisiae exhibits agrowth-suppressive phenotype. This is thought to be attributed to theactivation of the p68 kinase and subsequent phosphorylation of the yeastequivalent of mammalian elF2α (Chong et al.; Cigan et al. (1982) Proc.Natl. Acad. Sci. USA 86, 2784-2788).

[0012] The present inventor has surprisingly discovered by manipulatingthe expression of certain ISGs that manipulation of ISGs can havebeneficial uses. They have discovered that suppression of the expressionof the PKR protein or the 2-5A synthetase protein or both results in asubstantially higher viral yield from virus-infected cells which isuseful for enhancing the production of vaccines in animal cell culture.

Relevant Literature

[0013] A common approach to examine the biological role of PKR involvesthe generation of mutants deficient in the kinase activities. Since PKRpossesses a regulatory site for dsRNA binding and a catalytic site forkinase activity, investigators have used block deletion or site-directedmutagenesis to generate mutants at the regulatory or catalytic site. APKR dominant negative mutant, [Arg²⁹⁶]PKR, which contains a single aminoacid substitution of arginine for the invariant lysine in the catalyticdomain II at position 296 has been described (Visvanathan, K. V. &Goodbourne, S. (1989) EMBO J. 8, 1129-1138; D'Addario, M., Roulston, A.,Wainberg, M. A. & Hiscott, J. (1990) J Virol. 64, 6080-6089). Thismutant protein [Arg²⁹⁶]PKR can specifically suppress the activity ofendogenous wild-type PKR in vivo. Additional mutants have been generatedby altering the dsRNA binding motifs. For example, Feng et al. (ProcNatl Acad Sci USA 1992 89:5447-5451) abolished dsRNA binding ability ofPKR by deletional analysis to obtain mutants with deletions betweenamino acid residues 39-50 or 58-69. Similarly, other investigators havemutated amino acid residues in the N-terminal region to suppress dsRNAbinding ability leading to loss of PKR enzymatic activities (Green S R,Mathews M B. Genes & Development 1992 6:2478-2490; McCormack S J, OrtegaL G, Doohan J P, Samuels C E. Virology 1994 198:92-99). A recent articlehas further identified two amino acid residues that are absolutelyrequired for dsRNA binding, namely glycine 57 and lysine 60 (McMillan NA J, Carpick B W, Hollis B, Toone W M, Zamanian-Daryoush, and Williams BR G. J. Biol. Chem. 1995 270:2601-2606). Mutants in these positions wereshown to be unable to bind dsRNA in vitro and possessed noantiproliferative activity in vivo when expressed in murine macrophagecells.

[0014] The physiological significance of the loss of PKR activity invivo has been examined in animals. Catalytically inactive PKR mutants(including [Arg²⁹⁶]PKR) when transfected into NIH 3T3 (mouse fibroblast)cells caused suppression of endogenous PKR activity in thetransfectants. When administered to nude mice, these transfected cellscaused tumor formation suggesting a tumor suppressor activity for PKR(Koromilas, A. E., Roy, S., Barber, G. N., Katze, M. G. & Sonenberg, N.(1992) Science 257, 1685-1689; Meurs, E. F., Galabru, J., Barber, G. N.,Katze, M. G. & Hovanessian, A. G. (1993) Proc. Natl. Acad. Sci. USA 90,232-236). Meurs et al. (J. Virol. 1992 66:5805) produced stabletransfectants of NIH 3T3 cells with either a wild type (wt) PKR gene ora dominant negative mutant under control of a CMV promoter and showedthat only transfectants receiving the wt clone were partially resistantto infection with encephalomyocarditis virus (EMCV). Lee et al. (Virol.1993 193:1037) constructed a recombinant vaccinia virus vectorcontaining the PKR gene under control of an inducible promoter andshowed that in HeLa cells infected with the recombinant virus andinduced resulted in an inhibition of the vaccinia virus protein and anoverall decrease in viral yield. Henry et al. (J. Biol. Regulators andHomeostatic Agents 1994 8:15) showed that reoviral mRNAs containing aPKR activator sequence are poorly expressed in comparison with otherreoviral mRNAs but that addition of 2-aminopurine, a PKR inhibitor, ortransfection with a dominant negative PKR mutant, specifically increasedthe expression of mRNA containing the activator sequence. Maran et al.(Science 1994 265:789) showed that HeLa cells that were selectivelydeleted for PKR mRNA by treatment with PKR antisense oligos linked to2′-5′ oligoA were unresponsive to activation of nuclear factor-κB by thedsRNA poly(I):poly(C).

[0015] Several strategies have been utilized in the effort to improvethe yield of virus obtained from cell culture for vaccine production.Different cell types have been tested to obtain the best cell line foroptimum growth of specific viruses. The diploid human embryonic lungcell lines, MRC-5 and WI-38, have been developed specifically forvaccine production (see Pearson Devel. Biol. Standard. 1992 76:13-17;MacDonald, C. Critical Reviews Biotech. 1990 10:155-178; Wood et al.Biologicals 1990 18:143-146). Other attempts to improve vaccineproduction from cell culture include use of a low protein serumreplacement factor (Candal et al. Biologicals 1991 19:213-218), andtreatment of the cell culture with proteolytic enzymes (U.S. Pat. No. RE33,164).

SUMMARY OF THE INVENTION

[0016] It is an object of the present invention to provide a method forenhanced vaccine production in cell culture. It is another object of theinvention to provide methods for the evaluation of antiviral compoundsand for the identification and culture of viral pathogens.

[0017] These objects are generally accomplished by providing animal cellcultures in which the expression of the interferon genes issubstantially decreased from the normal level of expression. This may beeffected by manipulating the level of expression of factors thatfunction in vivo to regulate the interferon level, including interferontranscriptional regulators (for example, IRF1), interferon receptors andinterferon stimulated gene products (for example PKR and 2-5Asynthetase).

[0018] These objects are particularly accomplished by providing variousmethods using animal cell cultures in which the level ofinterferon-mediated antiviral protein activity, particularly fordouble-stranded RNA dependent kinase (PKR) and 2′-5′ Oligoadenylatesynthetase (2-5A synthetase), is significantly decreased from the normallevels. Among the various methods provided are methods for vaccineproduction, methods for determining the antiviral activity of acompound, and methods for detecting a virus in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIGS. 1A and 1B. PKR activity and protein levels in U937-derivedstable transfectant cell lines. FIG. 1 A Functional PKR activity wasdetermined using a poly(I):poly(C)cellulose assay for PKRautophosphorylation. Cell extracts were prepared from the different U937transfectant cell lines following incubation with (+) or without (−)recombinant human IFN-α2 (200 U/mL) as indicated, while L929 cells weresimilarly treated with mouse IFN-α/β. Lane 1, HeLa; lanes 2 and 3,U937-neo; lane 4, U937-AS1; lane 5, U937-AS3; lane 6, U937M13; lane 7,U937-M22; lane 8, L929. Positions of the human (68 kDa) and mouse (65kDa) PKR proteins, and the molecular size standards (80 and 50 kDa) areindicated. FIG. 1B Cell extracts were prepared as above after inductionwith IFN-α or -γ and PKR protein levels were determined by Western blotanalysis.

[0020]FIGS. 2A and 2B. Kinetics of EMCV replication are enhanced inPKR-deficient cells. The different U937 cell lines were challenged withEMCV at 0.1 (FIG. 2A) or 0.001 (FIG. 2B) TCID₅₀/cell. Samples wereharvested at the indicated times and viral yields were measured in termsof TCID₅₀.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0021] The present invention relies upon the discovery by the inventorthat the level of interferon production in cells can be regulated bymanipulating the expression or activity of certain factors that normallyregulate interferon expression and activity in vivo. These factorsinclude certain interferon-specific transcriptional regulators,particularly IRF1, certain interferon receptors, as well as the geneproducts of certain interferon simulated genes (also calledinterferon-mediated antiviral responses), particularly PKR and 2-5Asynthetase. Suppression or elimination of the expression or activity ofany of these factors will result in a lower than normal level ofexpression of interferon genes. One consequence of this lower thannormal interferon expression level is an increased permissiveness of thecell to viral replication. An increased permissiveness of the cell toviral reproduction means that greater viral production can be achievedin that cell relative to a cell having normal interferon expression.Cells having an increased permissiveness to viral replication are usefulfor a number of applications including vaccine production, sensitivedetection of low levels of virus and for the evaluation of antiviralcompounds.

[0022] The present inventor has surprisingly found that animal cellsthat are deficient in interferon-mediated antiviral responses,particularly cells deficient in dsRNA dependent kinase, 2 N-5 NOligoadenylate synthetase or both, produce a higher viral yield wheninfected with an animal virus than cells with normal levels of theseproteins. Increases of viral yield by as much as 10³ to 10⁴ or more canbe obtained using the method of the present invention. The ability toobtain high yields of virus in PKR- or 2-5A synthetase-deficient cellculture makes it possible to produce large amounts of virus within ashort time. This is particularly important for production of viralvaccines, most particularly for RNA virus, including influenza virus.The increased permissiveness of the deficient cells to viral replicationmakes them useful in a method for evaluating antiviral drugs in cellculture and in a method for detecting viral pathogens.

[0023] One aspect of the present invention provides a method forproduction of a viral vaccine in cell culture which comprises (a)infecting a cell culture with a donor strain animal virus, wherein saidcell culture is deficient in the activity of the gene product of aninterferon-stimulated gene, (b) culturing the infected cell cultureunder conditions sufficient to provide efficient virus growth, and (c)harvesting the virus produced. The harvested virus may be additionallyprepared for vaccine use by purification, for instance by sterilefiltration, ultrafiltration and/or concentration by columnchromatography or other methods. The harvested virus may optionally betreated to inactivate the virus for the production of killed viralvaccines.

[0024] In a preferred embodiment, the cell culture is deficient in PKRactivity. By PKR-deficient is meant that the PKR activity is less than5% of the normal level of PKR activity. By normal level of PKR activityis meant the PKR activity observed in the parental cell culture fromwhich the stable PKR-deficient cells are obtained or, if thePKR-deficiency is transiently induced, the PKR activity level observedin the cells before induction to PKR-deficiency. Preferably, thePKR-deficient cells have less than 1% of the normal level of PKRactivity, more preferably the PKR-deficient cells have less than 0.1% ofthe normal level of PKR activity. By PKR activity is meant the abilityto mediate the antiviral and antiproliferative activities of IFN-α andIFN-β, the ability to phosphorylate initiation factor elF-2α, or theability to phosphorylate IκBα to release nuclear factor κB. By PKR ismeant human p68 kinase or any analog or homolog of human p68 kinase. Byanalog of human p68 kinase is meant any double-stranded RNA-dependentkinase that mediates ds-RNA activation of interferon transcription.Typically, such ds-RNA dependent kinases are p68 kinase equivalentspresent in other species, such as, for example, rabbits or mice and indifferent tissues among the various species. For example, murine p65kinase is an analog of human p68 kinase. Another example of an analog ofp68 kinase has been described in human peripheral blood mononuclearcells (Farrel et al.) By homolog is meant a protein homologous to atleast one domain of human p68 kinase, such as, for example, thedsRNA-binding domain or the kinase domain. One such functional kinasehomolog is yeast GCN2 kinase.

[0025] PKR-deficient cells can be obtained by any of a variety ofmethods that are well-known in the art. PKR-deficient mutants can bestably PKR-deficient or may be transiently induced to PKR-deficiency.Techniques for producing stable PKR-deficient mutants include, but arenot limited to, random or site-directed mutagenesis (for example, Deng WP, and Nickoloff J A Analytical Biochemistry 1992 200:81-88; Busby S,Irani M, Crombrugghe B. J. Mol Biol 1982 154:197-209), targeted genedeletion (“gene knock-out”) (for example, Camper S A, et al. Biology ofReproduction 1995 52:246-257; Aguzzi A, Brandner S, Sure U et al. BrainPathology 1994 4:3-20), transfection with PKR antisense polynucleotides(for example, Lee et al. Virology 1993 192:380-385) and transfectionwith a PKR dominant negative mutant gene.

[0026] A PKR dominant mutant is a PKR mutant for which only a singleallele need be expressed in order to suppress normal PKR activity. PKRdominant mutant genes include a mutant human p68 kinase, a mutant murinep65 kinase, and mutants of any other ds-RNA dependent kinases or mutantsof analogs or homologs of human p68 kinase that suppress normal PKRactivity, for example [Arg²⁹⁶]PKR (Meurs et al. J. Virol. 199266:5805-5814). Examples of other PKR dominant mutants include mutants ofPKR obtained from rabbit reticulocytes, different mouse tissues andhuman peripheral blood mononuclear cells (Farrel et al., Levin et al.,Hovanessian, Krust et al., Buffet-Janvresse et al.) PKR dominant mutantsinclude mutants of functional homologs that suppress protein synthesisby interfering with initiation factor phosphorylation, particularlyphosphorylation of elF-2α. One such functional kinase homolog mutant isa mutant of yeast GCN2 kinase.

[0027] Techniques for producing cells that are transiently PKR-deficientinclude, but are not limited to, use of 2′-5′ oligoadenylate-linked PKRantisense oligonucleotides (Maran, A., Maitra, R. K., Kumar, A., Dong,B., Xiao, W., Li, G., Williams, B. R. G., Torrence, P. F. & Silverman,R. H. (1994) Science 265, 789-792) or specific inhibitors of the PKRprotein, such as 2-aminopurine (Marcus, P. I. & Sekellick, M. J. (1988)J Gen. Virol. 69, 1637-45, Zinn, K., Keller, A., Whittemore, L. A. &Maniatis, T. (1988) Science 240, 210-3) as well as other competitiveinhibitors that can block phosphorylation of PKR substrates, orinhibitors that can block double-stranded RNA binding. TransientlyPKR-deficient cell cultures can be obtained by culturing a cell line inthe presence of such antisense oligonucleotides or inhibitors.

[0028] Preferably for use in the method of the present invention, cellcultures will be stably PKR-deficient. Typically, PKR-deficient cellcultures are produced by transfection of a parent cell line, preferablya cell line currently used in vaccine production, preferably MRC-5,WI-38, or Vero (African Green Monkey cell), with a vector containing afunctional PKR antisense gene construct or a PKR dominant negativemutant construct followed by selection of those cells that have receivedthe vector. A functional PKR antisense gene construct may be prepared byconventional methods; for example, by cloning a PKR cDNA such as thatdescribed in Meurs et al. (Cell 1990 62:379-390), in an antisenseorientation, under the control of an appropriate promoter, for example aCMV promoter. A PKR dominant negative mutant construct can be preparedby cloning the cDNA for a PKR dominant negative mutant, for example thecDNA for [Arg²⁹⁶]PKR, under the control of an appropriate promoter.

[0029] Preferably the PKR mutant gene constructs are cloned under thecontrol of an inducible promoter to reduce the risk of tumor formationby these PKR-deficient cells since the cells are to be used for vaccineproduction in the methods of the invention. This method will ensure thesafety of the vaccines produced by these cells. The loss of PKR activityhas been associated with tumor formation (Koromilas et al.; Meurs etal.). Although the harvested virus can be purified from cell culturecomponents, there nevertheless remains a risk that some PKR-deficientcells would be carried over into the final vaccine preparation. If PKRactivity remains constitutively suppressed, these cells may potentiallybecome tumorigenic. This would create potential health risk for thevaccine recipient. However, if an inducible promoter is used to controlexpression of the gene construct, endogenous PKR activity would berestored upon removal of the inducer. Suitable inducible promotersinclude a lac promoter, a heat shock promoter, a metallothioneinpromoter, a glucocorticoid promoter, or any other inducible promoterknown to one skilled in the art.

[0030] Other ways of constructing similar vectors, for example usingchemically or enzymatically synthesized DNA, fragments of the PKR cDNAor PKR gene, will be readily apparent to those skilled in the art.Transfection of the parental cell culture is carried out by standardmethods, for example, the DEAE-dextran method (McCutchen and Pagano,1968, J. Natl. Cancer Inst. 41:351-357), the calcium phosphate procedure(Graham et al., 1973, J. Virol. 33:739-748) or by any other method knownin the art, including but not limited to microinjection, lipofection,and electroporation. Such methods are generally described in Sambrook etal., Molecular Cloning: A laboratory manual, 2nd Edition, 1989, ColdSpring Harbor Laboratory Press. Transfectants having deficient PKRactivity are selected. For ease of selection, a marker gene such asneomycin phosphotransferase II, ampicillin resistance or G418resistance, may be included in the vector carrying the antisense ormutant gene. When a marker gene is included, the transfectant may beselected for expression of the marker gene (e.g. antibiotic resistance),cultured and then assayed for PKR activity.

[0031] Residual PKR activity in PKR-deficient cells can be determined byany of a number of techniques that are well-known in the art. Theactivity of PKR can be determined directly by, for example, anautophosphorylation assay such as that described in Maran et al.(Science 265:789-792 1994) or Silverman et al. (Silverman, R. H., andKrause, D. (1986) in Interferons: A practical approach. Morris, A. G.and Clemens, M. J., eds. pp. 71-74 IRL Press, Oxford-Washington, D.C.).Typically, an autophosphorylation assay for PKR activity is carried outas follows. Extracts from cells to be examined for PKR activity whichcontain approximately 100 μg of protein are incubated with 20 μl ofpoly(I):poly(C)-cellulose beads for 60 min on ice. The kinase isimmobilized and activated on the beads. After washings of thepolynucleotide cellulose-bound kinase fractions, an autophosphorylationreaction is performed at 30° C. for 30 min in an assay solution. Theassay solution contains 1 μCi of [γ³²P]ATP, 1.5 mM magnesium acetate, 10μM ATP pH 7.5, 0.5% NP 40, and 100 μg/ml leupeptin. The samples areheated at 90° C. for 3 min in gel sample buffer containing sodiumdodecyl sulfate (SDS) and the proteins are analyzed by 10%SDS-polyacrylamide gel electrophoresis. The gels are dried andautoradiographs are prepared using XAR-5 X-ray film (KodaK).

[0032] Residual PKR activity may also be determined indirectly byassaying for the presence of the PKR protein, for example by Westernblot with PKR specific antibodies, or for the presence of PKR RNA, forexample by Northern blot with oligonucleotide or cDNA probes specificfor PKR. As will be readily apparent, the type of assay appropriate fordetermination of residual PKR activity will in most cases depend on themethod used to obtain the PKR-deficient phenotype. If, for example, themethod used to produce the PKR-deficient cell results in suppression orelimination of PKR gene expression (for example, gene knock-out),analysis techniques that detect the presence of mRNA or cDNA (e.g.Northern or Southern blots) or the presence of the protein (e.g. Westernblot) or that detect the protein activity may be useful to determine theresidual PKR activity in the PKR-deficient cells. On the other hand, ifthe method used to produce the PKR-deficient cells results in inhibitionof the protein rather than elimination of expression of the gene (forinstance, transfection with a vector carrying a dominant negative PKRmutant), an autophosphorylation assay is more appropriate than a Westernblot for determination of the residual PKR activity.

[0033] In another embodiment, the present invention provides a methodfor production of a viral vaccine in a cell culture that is deficient in2′-5′Oligoadenylate synthetase activity. A cell culture deficient in2-5A synthetase can be isolated in a similar fashion to cell culturesdeficient in PKR, for example, random or site-directed mutagenesis,targeted gene deletion of the 2-5A synthetase genes or transfection withantisense 2-5A synthetase constructs. By 2-5A synthetase-deficient ismeant that the 2-5A synthetase activity is less than 5% of the normallevel of 2-5A synthetase activity. By normal level of 2-5A synthetaseactivity is meant the 2-5A synthetase activity observed in the parentalcell culture from which the stable 2-5A synthetase-deficient cells areobtained or, if the 2-5A synthetase-deficiency is transiently induced,the 2-5A synthetase activity level observed in the cells beforeinduction to 2-5A synthetase-deficiency. Preferably, the 2-5Asynthetase-deficient cells have less than 1% of the normal level of 2-5Asynthetase activity, more preferably the 2-5A synthetase-deficient cellshave less than 0.1% of the normal level of 2-5A synthetase activity.Residual 2-5A synthetase activity in 2-5A synthetase-deficient cells canbe determined by methods similar to those used for determining residualPKR activity, that is, Western blots using 2-5A synthetase specificantibodies, Northern blots using oligonucleotide or cDNA probes specificfor 2-5A synthetase or enzyme activity assays (see, Read et al. J.Infect. Dis. 1985 152:466-472; Hassel and Ts'o J. Virol. Methods 199450:323-334). Typically, 2-5A synthetase activity is determined asfollows. Cells to be assayed are treated with IFN-α₂ (100 U/lml in RPMIplus 10% fetal bovine serum). Briefly, the cell cultures are incubatedfor 18 hr at 37° C., washed and the cell pellets are treated with celllysis buffer for 10 min at 4° C. Aliquots of the cellular extract areincubated with poly(I):poly(C)agarose beads for 30 min at 30° C., toallow for binding as well as activation of the 2-5A synthetase enzyme.The beads are washed and then incubated in an assay solution containing3 mM ATP, 4 μCi ³H-ATP per assay sample, and 20 mM Hepes buffer pH 7.5for 20 hr at 30° C. Following incubation, the samples are heated at 90°C. to inactivate the enzyme, followed by treatment with bacterialalkaline phosphatase (BAP). The 2-5 oligoA synthesized is resistant toBAP. The amount of 2-5 oligo A is determined by spotting a sample ontofilter paper, washing and counting the ³H radioactivity using ascintillation counter. The amount of oligoA product produced iscorrelated with the enzyme activity by conventional methods.Alternatively, 2-5A synthetase can be assayed by a radioimmune andradiobinding method (Knight M, et al. Radioimmune, radiobinding and HPLCanalysis of 2-5A and related oligonucleotides from intact cells Nature1980 288:189-192).

[0034] It will be apparent that cell cultures deficient in both PKRactivity and 2-5A synthetase activity can be made by a combination ofthe methods described above. The doubly deficient cell cultures can beprepared either sequentially (that is, by first selecting culturesdeficient in one activity and then using that cell culture as thestarting material for preparing the second deficient culture) orsimultaneously (selection for both deficiencies at once).

[0035] In another embodiment, the present invention provides a methodfor production of a viral vaccine in a cell culture that is deficient inhuman MxA protein activity. A cell culture deficient in human MxAprotein activity can be isolated in a similar fashion to cell culturesdeficient in PKR, for example, random or site-directed mutagenesis,targeted gene deletion of the MxA genes or transfection with antisenseMxA constructs. By MxA protein-deficient is meant that the MxA activityis less than 5% of the normal level of MxA activity. By normal level ofMxA activity is meant the MxA activity observed in the parental cellculture from which the stable MxA-deficient cells are obtained or, ifthe MxA-deficiency is transiently induced, the MxA activity levelobserved in the cells before induction to MxA-deficiency. Preferably,the MxA-deficient cells have less than 1% of the normal level of MxAactivity, more preferably the MxA-deficient cells have less than 0.1% ofthe normal level of MxA activity. Residual MxA activity in MxA-deficientcells can be determined by methods similar to those used for determiningresidual PKR activity, that is, Western blots using MxA specificantibodies, Northern blots using oligonucleotide or cDNA probes specificfor MxA or enzyme activity assays (Garber et al. (1991) Virology 180,754-762; Zurcher et al. (1992) Journal of Virology 66, 5059-5066).Typically, MxA activity is determined as described in Zürcher et al.

[0036] In yet another embodiment, the present invention provides amethod for production of a viral vaccine in a cell culture that isdeficient in interferon responsiveness. By interferon responsiveness ismeant the ability of a cell to respond to stimulation by interferon. Acell culture deficient in interferon responsiveness can be obtained byculturing the cells in the presence of an inhibitor of an interferonreceptor. Alternatively, cells can be engineered to express, in theabsence of a normal interferon receptor, a mutant interferon receptorthat is unresponsive to interferon.

[0037] In another embodiment, the present invention provides a methodfor production of a viral vaccine in a cell culture that is deficient ininterferon-specific transcriptional regulators. One suchinterferon-specific transcriptional regulator is IRF1. Cells stablydeficient in interferon-specific transcriptional regulators can beobtained by any of a number of techniques well known in the art, suchas, for example, random or site-directed mutagenesis, targeted genedeletion, or transfection with antisense vectors. Transiently deficientcells can be obtained by culturing cells in the presence of antisenseoligonucleotides or specific inhibitors of interferon transcription.

[0038] The method of the present invention can be practiced with avariety of animal cell cultures, including primary cell cultures,diploid cell cultures and continuous cell cultures. Particularly usefulare cell cultures that are currently used for the production of vaccine,most particularly those cell cultures that have been approved forvaccine production by the USFDA and or WHO, for example, MRC-5, a humandiploid cell line from fetal lung tissue (Nature Lond. 1970 227:168-170)and WI-38, a human diploid cell line derived from embryonic lung tissue(Am. J. Hyg. 1962 75:240; First International Conference on VaccinesAgainst Viral and Rickettsial Diseases of Man, Pan American HealthOrganization, Pub. No. 147: 581 May 1981). Also useful are Chang livercells (Chang, R S Proc. Soc. Exp. Biol. Med. 1954 87:440), U937 humanpromonocytic cells (Sundstrom et al. Int. J. Cancer 1976 17:565-577),Vero cells, MRC-9 cells, 1MR-90 cells, 1MR-91 cells and Lederle 130cells (Biologicals 18:143-146 1991). U937 cells are particularly usefulfor viruses that infect immune cells expressing CD4, for example, HIV.For a general review of cell cultures used in the production of vaccinessee Grachev, V. P. in Viral Vaccines Mizrahi, A. ed. pages 37-67 1990Wiley-Liss. The particular cell culture chosen will depend on the viruswhich is to be produced; in general, the cell culture will be derivedfrom the species which is the natural host for the virus, although thisis not essential for the practice of the present invention (for example,human virus can be grown on a canine kidney cell line (MDCK cells) or agreen monkey kidney cell line (Vero cells; Swanson et al. J. Biol.Stand. 1988 16:311)). Typically, the cells chosen will be PKR-deficientor 2-5A synthetase-deficient derivatives of cells or cell lines known tobe an appropriate host for the virus to be produced. For example, forinfluenza virus and hepatitis A virus vaccines, preferred host cells arederivatives of MRC-5. For HIV vaccine production, preferred host cellsare derivatives of U937, H9, CEM or CD4-expressing HUT78 cells. Celllines used for the production of vaccines are well known and readilyavailable from commercial suppliers, for example, American Type CultureCollection.

[0039] The infection of the interferon-mediated antiviralresponse-deficient cells with donor virus according to the presentinvention is carried out by conventional techniques (see for examplePeetermans, J. Vaccine 1992 10 supp 1:S99-101; Shevitz et al. in ViralVaccines Mizrahi, a. ed. pp 1-35 1990 Wiley-Liss). Typically, virus isadded to the cell culture at between 0.001 to 0.5 TCID₅₀ per cell,preferably at 0.01 to 0.10 TCID₅₀ per cell, but will vary as appropriatefor the particular virus and cell host being used. As is readilyapparent to one of ordinary skill in the art, every cell of the cellculture need not be infected initially for efficient viral production.The infected cells are cultured under conditions appropriate for theparticular cells and viral production at various times after infectionis monitored. Viral production can be monitored by any of a number ofstandard techniques including plaque-forming unit assays, TCID₅₀ assaysor hemagglutination inhibition assays (Robertson et al. J. Gen. Virol.1991 72:2671-2677). The infected cells are cultured under conditionssufficient to provide efficient viral growth. The cells can be cultureduntil maximum viral production is achieved as indicated by a plateauingof the viral yield. The virus is harvested by standard techniques andsubstantially purified from other cellular components (see for example,Peetermans 1992). The harvested virus may be used as a live viralvaccine, either fully virulent or attenuated, or may be inactivatedbefore use by methods that are well-known in the art, for example, bytreatment with formaldehyde (Peetermans, J Vaccine 1992 10 Suppl1:S99-101; U.S. Pat. No. RE 33,164).

[0040] The vaccine may be available in dry form, to be mixed with adiluent, or may be in liquid form, preferably in aqueous solution,either concentrated or ready to use. The vaccine is administered aloneor in combination with pharmaceutically acceptable carriers, adjuvants,preservatives, diluents and other additives useful to enhanceimmunogenicity or aid in administration or storage as are well-known inthe art. Suitable adjuvants include aluminum hydroxide, alum, aluminumphosphate, Freunds or those described in U.S. Pat. Nos. 3,790,665 and3,919,411. Other suitable additives include sucrose, dextrose, lactose,and other non-toxic substances. The vaccines are administered to animalsby various routes, including intramuscular, intravenous, subcutaneous,intratracheal, intranasal, or by aerosol spray and the vaccines arecontemplated for the beneficial use in a variety of animals includinghuman, equine, avian, feline, canine and bovine.

[0041] The method of the present invention can be practiced with avariety of donor animal viruses. By donor virus is meant the particularviral strain that is replicated in vitro to produce the vaccine. Theparticular donor animal virus used will depend upon the viral vaccinedesired. Donor viruses currently used for vaccine production arewell-known in the art and the method of the present invention can bereadily adapted to any newly identified donor virus. Preferred donorviruses include human influenza virus, especially influenza A (H3N2) andinfluenza A (H1N1) (see U.S. Pat. No. 4,552,758; ATCC Nos. VR-2072,VR-2073, VR-897); influenza A described in U.S. Pat. No. 3,953,592;influenza B (U.S. Pat. No. 3,962,423; ATCC Nos. VR-786, VR-791); andParainfluenza 1 (Sendai virus) (Cantell et al. Meth. Enzymol.78A:299-301 1980; ATCC No.VR-907). The donor virus can be identical tothe viral pathogen or may be a naturally-occurring attenuated form, anattenuated form produced by serial passage through cell culture or arecombinant or reassortant form. Any viral strain may be used as donorvirus provided that it retains the requisite antigenicity to affordprotection against the viral pathogen. The method of the presentinvention is particularly useful with attenuated or poorly replicatingdonor viruses.

[0042] Some of the vaccines that can be provided by the methods of thepresent invention include, but are not limited to, human vaccines forpoliovirus, measles, mumps, rubella, hepatitis A, influenza,parainfluenza, Japanese encephalitis, cytomegalovirus, HIV, Dengue fevervirus, rabies and Varicella-zoster virus, as well as many non-humananimal vaccines including, for example, vaccines for feline leukemiavirus, bovine rhinotracheitis virus (red nose virus), cowpox virus,canine hepatitis virus, canine distemper virus, equine rhinovirus,equine influenza virus, equine pneumonia virus, equine infectious anemiavirus, equine encephalitis virus, ovine encephalitis virus, ovine bluetongue virus, rabies virus, swine influenza virus and simianimmunodeficiency virus. As will be apparent from the foregoing, themethod of the present invention is not limited to vaccine production forhuman viruses but is equally suitable for production of non-human animalviral vaccines.

[0043] Another aspect of the present invention provides a method forevaluating the activity of antiviral compounds. Due to the increasedpermissiveness of the PKR-deficient cells to viral replication, thecells are useful in a sensitive assay for assessing the effectiveness ofantiviral compounds. In this aspect, the present invention comprises thesteps of (a) treating a virus, virus-infected host cells or host cellsprior to virus infection with the antiviral compound and (b) assayingfor the presence of remaining infectious virus by exposure underinfective conditions of a PKR-deficient or 2-5A synthetase-deficientindicator cell culture.

[0044] In this aspect, the virus against which the antiviral compound isto be tested may be treated directly with the compound. In this case,the treated virus may then be analyzed directly for the presence ofremaining infectious virus by exposure under infective conditions of aPKR-deficient or 2-5A synthetase-deficient indicator cell culture to analiquot of the treated virus, culturing for a time sufficient to allowreplication of any remaining infectious virus and analyzing theindicator culture for the presence of the replicated virus.Alternatively, the virus against which the antiviral compound is to betested may be used to infect a host cell culture, the infected host cellculture is then treated with the antiviral compound. A cell extract ofthe treated infected host cell culture is prepared by conventionaltechniques and an aliquot of the extract is analyzed for the presence ofremaining infectious virus by exposure to a PKR-deficient or 2-5Asynthetase-deficient indicator cell culture as described above. Inanother alternative, the host cell culture may be treated with theantiviral compound prior to infection with the virus rather than afterinfection. The treated cells are then infected with the virus againstwhich the antiviral compound is to be tested, cultured and analyzed forthe presence of replicated virus. The particular treatment regime chosenwill depend upon the known or postulated mode of action of the antiviralcompound and will be readily within the determination of one skilled inthe art. By exposure under infective conditions is intended the bringingtogether the deficient indicator cell culture and an aliquot of thetreated sample (either virus or infected cell extract) under conditionsthat would result in infection of the deficient cell culture if anyvirus was present in the treated sample. After exposure to the treatedsample, the deficient indicator cell culture is cultured further andassayed for the replication of the virus, by standard method (forexample, plaque assays or TCID₅₀assays or Northern or Western analysisfor viral RNA or protein).

[0045] The host cell culture may be any cell culture which issusceptible to infection by the virus against which the antiviralcompound is to be tested. The indicator cell culture is a PKR-deficientor 2-5A synthetase deficient cell culture that is used to assay forinfectious virus remaining after treatment with the antiviral compound.The indicator PKR-deficient or 2-5A synthetase deficient cell culture isprepared as described above for vaccine production. Cells suitable as aparent for generating the deficient indicator are the same as those thatare useful for generating the PKR-deficient or 2-5A synthetase deficientcell cultures for vaccine production. In addition, the following celllines are also suitable: hepatoma cell lines in general, particularlyHep G2 human hepatocellular carcinoma (Nature 1979 282:615-616; U.S.Pat. No. 4,393,133) and Hep 3B (U.S. Pat. No. 4,393,133). It will beapparent that the indicator cell culture is also susceptible toinfection by the virus against which the antiviral compound is to betreated. The host cell culture and the indicator cell culture may be thesame or different. The antiviral compound can be any chemical orbiological preparation suspected of having some antiviral activity. Ifthe virus itself is treated with the antiviral compound, the compoundmay be removed before infection of the indicator cell culture byexposure to the treated virus. If an infected host cell culture (or apre-infected host cell culture) is treated with the antiviral compound,the compound may be removed before preparation of the cell extract.

[0046] In a separate related aspect, the present invention provides amethod for identification and culture of viral pathogens. Thepermissiveness of PKR-deficient cells to viral replication makes themparticularly useful in a method to detect very low levels of virusand/or viruses that are difficult to culture, for example, HIV inmonocytes or lymphocytes of neonates. In this aspect the presentinvention comprises the steps of (1) exposing under infective conditionsa PKR-deficient or a 2-5A synthetase-deficient cell culture to a samplesuspected of containing a virus and (2) assaying for the presence ofreplicated virus in the exposed cells. The practice of this aspect ofthe present invention is similar to that of the previous aspect exceptthat treatment with antiviral compound is omitted. In this aspect, thesample to be assayed for the presence of virus is generally a clinicalsample from a patient suspected of having a viral infection. The samplemay be any appropriate clinical sample including blood, saliva, urine,as well as biopsy samples from lymph node, lung, intestine, liver,kidney and brain tissue. The sample may be treated appropriately torelease viral particles (for example, cell extracts may be prepared) orthe sample may be used as received from the patient. The sample or analiquot of the sample is exposed under infective conditions to adeficient indicator cell culture and the presence of any replicatingvirus is determined as described above.

[0047] Specific examples of the steps described above are set forth inthe following examples. However, it will be apparent to one of ordinaryskill in the art that many modifications are possible and that theexamples are provided for purposes of illustration only and are notlimiting of the invention unless so specified.

EXAMPLES Example 1 Preparation of Plasmids

[0048] The cDNA inserts corresponding to the wild type human PKR geneand the dominant negative [Arg²⁹⁶]PKR mutant gene, from the plasmidspBS-8.6R and yex6M (Meurs E, Chong K, Galabru J. et al. Cell 199062:379-90; Chong et al. EMBO J. 11:1553-1562 1992), respectively, werereleased by HindIII digestion and subcloned into pRC-CMV (Invitrogen), aconstitutive eukaryotic expression plasmid containing a G418-resistancemarker. The orientation of the inserts in selected clones was determinedby restriction digest analysis and confirmed by sequencing (Sequenase2.0, USB). This procedure resulted in the isolation of the expressionplasmids used, pPKR-AS (containing the PKR cDNA in an antisenseorientation under the control of the CMV promoter in the vector) andp[Arg²⁹⁶]PKR (containing the Arg²⁹⁶PKR cDNA under the control of the CMVpromoter in the vector).

Example 2 Isolation of PKR-deficient Stable Transfectants

[0049] Stable transfectants were obtained by electroporation of 5×10⁶exponentially growing U937 cells with 10 μg of each plasmid, inserum-free RPMI-1640 containing DEAE-dextran (50 μg/mL), with a GenePulser apparatus (BioRad) set at 500 μF, 250 V. Bulk populations ofstable transfectants were obtained by selection with 400 μg/mL geneticin(GIBCO-BRL) for 3 weeks. Clonal lines were subsequently obtained bylimiting dilution cloning. Cell lines were cultured in RPMI-1640containing 10% fetal calf serum (complete media) and geneticin.

[0050] Five representative cell lines were selected for initialcharacterization: “U937neo” (also called U9K-C) was the control cellline transfected with the parental vector, pRC-CMV; “U937-AS1” (alsocalled U9K-A1) and “U937-AS3”(also called U9K-A3) were independentclones transfected with pPKR-AS; “U937-M13” (also called U9K-M13) and“U937-M22” (also called U9K-M22) were independent clones transfectedwith p[Arg²⁹⁶]PKR.

Example 3 Characterization of PKR-deficient Transfectants

[0051] PKR kinase activity was measured in an autophosphorylation assaythat uses poly(I):poly(C)-cellulose for binding and activation of PKRenzyme. PKR autophosphorylation assay was performed essentially asdescribed by Maran et al. with the following modifications. Cellextracts (100 μg of protein per assay) were incubated withpoly(I):poly(C)-cellulose for 1 hour on ice, washed three times, andincubated for 30 minutes at 30° C. in 50 μl of a reaction buffer (20 mMHEPES (pH 7.5), 50 mM KCI, 5 mM 2-mercaptoethanol, 1.5 mM Magnesiumacetate, 1.5 mM MnCl₂) containing 1 μCi of [γ-³²P]ATP. Proteins wereseparated on a 10% SDS-polyacrylamide gel and analyzed byautoradiography.

[0052] Cell extracts from IFN-treated HeLa and mouse L929 cells wereused as positive controls, since PKR activity in these cells has beenpreviously characterized (Meurs et al.) (FIG. 1A, lanes 1 and 8).U937-neo cells contained low basal levels of PKR activity whichincreased following treatment with IFN-α (FIG. 1A, lanes 2 and 3). PKRactivity in the parental, untransfected U937 cells was similar toU937-neo cells. However, PKR activity was not detected in any of thefour cell lines transfected with pPKR-AS or p[Arg²⁹⁶]PKR plasmids.Furthermore, treatment of these cells with IFN-α did not restore PKRactivity (FIG. 1A, lanes 47), nor did treatment with IFN-γ.

Example 4 Western Analysis of PKR-deficient Transfectants

[0053] To further confirm the inhibition of PKR expression in thepPKR-AS-transfected cell lines, Western blot analysis was performedusing a monoclonal antibody specific for human PKR. Cell extracts (100μg) were separated on a 10% SDS-polyacrylamide gel andelectrotransferred onto nitrocellulose membrane. The membranes wereincubated with anti-PKR monoclonal antibody (Meurs et al. Cell 1990) at1:1000 in BLOTTO (5% nonfat dry milk, 0.05% Tween-20 in Tris-bufferedsaline). Final detection of PKR was facilitated by probing with asecondary horseradish peroxidase-conjugated goat anti-mouse antibody(Santa Cruz Biotech) and using a chemiluminesence method (Amersham ECL).

[0054] Basal level of PKR protein was detectable in U937-neo cells (FIG.1B, lane 1) and increased following treatment with IFN-α or IFN-γ (FIG.1B, lanes 2 and 3). In contrast, PKR expression was significantlydiminished in both U937-AS1 and U937-AS3 cells (FIG. 1B, lanes 4 and 6)and did not increase following treatment with IFN-α (FIG. 1B, lanes 5and 7). While PKR protein was detectable in U937-M13 and U937-M22 cells,the mutant [Arg²⁹⁶]PKR protein was not distinguishable from wild typePKR by using Western blot analysis.

Example 5 Enhanced EMCV Replication in PKR-deficient Cells

[0055] Since the IFN system plays a major role in antiviral responses,we investigated whether loss of PKR function would affect the rate ofencephalomyocarditis virus (EMCV) replication. Stocks of EMCV (ATCC No.VR-1314) were prepared by passage in L929 cells. For determination ofEMCV replication, U937-derived transfectants were cultured in completemedia with or without IFNs (recombinant human IFN-α2, Schering;recombinant human IFN-γ, Amgen) for 18 hours. Following two washingswith PBS, the cells were incubated with EMCV in serum-free media for 2hours. The cells were washed again twice and replenished with mediacontaining 1% FCS. Samples were collected at the required time pointsand lysed by three rounds of freeze-thaw. Four-fold serial dilutions ofthe samples were added onto L929 monolayers and incubated for 48 hours,followed by staining with 0.05% crystal violet to determine cytopathiceffects and median tissue culture infective dose (TCID₅₀).

[0056] In the control U937-neo cell line following challenge with EMCVat 0.1 TCID₅₀/cell, viral titers peaked at approximately 10⁴ TCID₅₀/mLafter 48 hours and did not increase further after 72 hours (FIG. 2A).However, in U937-AS1 and U937-M22 cells, EMCV replication wassubstantially higher reaching titers of 10⁴ to 10⁵ TCID₅₀/mL after only24 hours and 10⁸ TCID₅₀/mL by 48 hours, representing a 10³ to 10⁴increase in viral yield over that obtained in control cells. In separateexperiments using a lower virus inoculum of 0.001 TCID₅₀/cell, moredramatic differences were observed in EMCV susceptibility between thecontrol and the PKR-deficient cells (FIG. 2B). Under these conditions,EMCV replication in U937-neo cells was minimal, not exceeding 10²TCID₅₀/mL even after 72 hours, while high viral titers of 10⁸ TCID₅₀/mLwere attained after 48 hours in both U937-AS1 and U937-M22 cells. Theresults indicated that by suppressing PKR activity in vivo, the cellsbecome very permissive to viral replication, showing as much as athousand-fold increase over control cells.

[0057] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0058] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of thedescribed invention.

What is claimed is:
 1. A method for production of a viral vaccinecomprising: (a) selecting a cell used for the production of a vaccine;(b) making said cell deficient, wherein said deficient cell comprises atleast one interferon-stimulated gene and is deficient in the activity ofthe gene product of at least one of said interferon-stimulated genes;(c) infecting said deficient cell with a donor strain animal virus; (d)culturing said infected cell under conditions providing for efficientvirus growth; (e) harvesting the virus produced; and (f) preparing saidviral vaccine from said harvested virus for administration to humans. 2.The method of claim 1 , wherein said cell is deficient in PKR activityand said PKR-deficient cell is obtained by transfection of a parent cellline with a PKR dominant negative mutant gene.
 3. The method of claim 2, wherein said dominant negative mutant is a mutant of murine p65kinase.
 4. The method of claim 2 , wherein said dominant negative mutantis a mutant of PKR obtained from rabbit reticulocytes.
 5. The method ofclaim 2 , wherein said dominant negative mutant is a mutant of PKRobtained from human peripheral blood mononuclear cells.
 6. The method ofclaim 2 , wherein said dominant negative mutant is a mutant of yeastGCN2 kinase.
 7. The method of claim 1 , wherein said deficient cell isobtained by culturing a cell line in the presence of an inhibitor of thePKR protein.
 8. The method of claim 1 , wherein said deficient cell isdeficient in both PKR and 2-5A synthetase.
 9. The method of claim 1 ,wherein said deficient cell is a human cell.
 10. The method of claim 1 ,wherein said deficient cell is selected from the group of MRC-5, WI-38,Chang liver, U937, Vero, MRC-9, IMR-90, IMR-91, Lederle 130, MDCK, H9,CEM, and CD4-expressing HUT78.
 11. The method of claim 10 , wherein saiddeficient cell is a MRC-5 or WI-38 or Vero cell.
 12. The method of claim1 , wherein said deficient cell is a U937 cell.
 13. The method of claim1 , wherein said donor virus is an attenuated virus.
 14. The method ofclaim 1 , wherein said donor virus is a recombinant virus.
 15. Themethod of claim 1 , wherein said donor virus is a human virus.
 16. Themethod of claim 15 , wherein said donor virus is a human influenzavirus.
 17. The method of claim 1 , wherein said donor virus is anon-human virus.
 18. The method of claim 2 , wherein said dominantnegative mutant is a mutant of human p68 kinase.
 19. The method of claim1 , wherein said deficient cell is obtained by targeted gene deletion ofat least one of said interferon-stimulated genes.
 20. The method ofclaim 19 , wherein at least one of said interferon-stimulated genesencodes PKR.
 21. The method of claim 1 , wherein said virus isinactivated.
 22. The method of claim 1 , wherein said harvested virus ispurified.